1. Field of the Invention
The present invention generally relates to detection systems and methods and more particularly to improved systems and methods for identifying and determining the concentration of viruses.
2. Prior Art
Viruses constitute a group of infectious agents which are separately identifiable from other infectious microorganisms on the basis of their structure and their mode of replication and antigenicity. Viruses are the smallest infectious agents (20-300 mu. in diameter) containing a molecule of nucleic acid as their genome. The nucleic acid is encased in a protein shell and the entire infectious unit is called a virion. Virions replicate only in living cells. The viral nucleic acid contains information necessary for programming the infected host cell to synthesize a number of specific macro-molecules. Towards the end of this replication cycle more viral nucleic acid and coat proteins are produced, the latter assembling together to form the capsid which encases and stabilizes the viral nucleic acid and facilitates the attachment and penetration of the virus upon contact with new susceptible cells. Viral antiserum neutralizes the virus because it reacts with the antigens of the protein coat.
When a virus infects living animals, specific antibodies are formed and are recoverable in blood serum from the living host. The specific antibodies render the host immune to the antibodies being known as antiserum. If antiserum specific to a given virus is present in a sufficient amount in a test sample which also contains the given virus and tissue cells which can be attacked by that virus, the virus attack is prevented or neutralized by the specific antiserum, thus identifying the virus. Tests based on this concept in determining the identity of viruses are called neutralization tests. Various other serological tests have been devised and are named according to the nature of the changes observed as a result of contacting the antiserum with the virus, i.e., the antibody-antigen reaction. Thus, such tests are called agglutination, precipitation-flocculation tests, and hemagglutination-inhibiting and complement fixation tests, among others. Neutralization tests have proven to be particularly valuable in the identification of viruses.
In the usual types of neutralization tests, the virus to be identified is contacted with various specific antisera of known types, as well as normal serum. Tissue cells are placed into contact with the virus and serum mixtures. After incubation of the test cells plus virus and serum, the cytopathic effect, if any, of the virus on the cells is determined microscopically and indicates the nature of the virus. The cytopathic effect will vary according to the type of cell and the type of virus. Typically, tissue cells when infected by virus exhibit a change in physical shape. Thus, for example, they may tend to become round rather than irregular or they may clump together and/or shrink. If the antiserum present in a given test sample neutralizes the effect of the virus, then the virus is identifiable as being of the same type as caused the elaboration of the antibodies in the antiserum.
In performing a neutralization test, it is usual to first run a series of titers in order to determine the approximate concentration of the virus being tested. This is because most such tests require the use of a known concentration of virus, for example 100 CPED.sub.50. CPED.sub.50 means the virus dose which will produce cytopathic effects on test tissue cells in 50% of the cases. In order to determine what dilution of the virus will bring it to a concentration of 100 CPED.sub.50, a calculation is usually made, based on the titer results, according to the method of Reed & Muench (American Journal of Hygiene 27:493-497, 1938).
Normally, a considerable period of time is required in order to carry out the necessary titration procedure so as to determine the concentration of the virus and thus be able to adjust it to 100 CPED.sub.50. Further time is required to test the virus in 100 CPED.sub.50 concentration against various dilutions of various antisera to determine which particular virus is involved. Moreover, substantial amounts of reagents and laboratory equipment and a plurality of skilled personnel are normally involved.
Ways have been sought to improve virus neutralization test procedures so as to reduce their steps and the time, personnel and equipment for carrying them out, thereby reducing their overall cost. Some of the newer neutralization tests employ micro-titration plates which contain a plurality of titration wells for simultaneously running parallel tests. However, these wells must be filled through micro-pipettes with various concentrations of antisera and virus isolates, and in some cases normal sera, in order to perform the test procedures. It has been found that it is relatively difficult, particularly when laboratory time is in short supply, to utilize the micro-titration plates because great care must be taken in transferring the liquid antisera and isolates to the wells to assure their proper dilutions. This is particularly the case when up to 96 or more wells are used in a single micro-titration plate. Attempts to automate such tests have substantially increased costs due to complicated automation equipment and necessary equipment clean-up time for skilled, high salaried personnel.
Accordingly, there remains a need for improved means and procedures for carrying out virus neutralization tests economically, rapidly and efficiently with accuracy.